■慢性HBV感染不能通过目前的治疗方法治愈,因为它们在感染个体的肝脏中减少共价闭合环状(ccc)DNA水平的能力有限。因此,更多的cccDNA的形成和持久性的分子决定因素的理解是必要的。一个关键问题是从头核衣壳介导的补充(再进口)对感染肝细胞中cccDNA水平的贡献程度。
■我们设计了一种感染性HBV突变体,其基因组在HBV核心开放阅读框(ΔHBcHBV)的T67位置编码终止密码子。重要的是,ΔHBcHBV病毒粒子在感染时不能启动核衣壳合成。长期在体外HBV感染标志物随访9周HepG2-NTCP细胞(A3克隆)和HBVDNA定量使用新开发的,高精度PCR测定(cccDNA倒置定量PCR)。
■ΔHBc和野生型(WT)HBV导致HBV表面抗原(HBsAg)的可比表达,可以用进入抑制剂MyrcludexB阻断,通过受体牛磺胆酸钠协同转运多肽(NTCP)确认真正的感染。在原代人肝细胞中,Huh7-NTCP,HepG2-NTCP,和HepaRG-NTCP细胞,形成相当的cccDNA拷贝数。cccDNA水平,病毒RNA的转录,和HBsAg分泌在WT和ΔHBcHBV感染细胞中保持相对稳定至少9周。
■我们的结果表明,从头合成的HBc在cccDNA的转录调控中起着次要作用。重要的是,我们表明,最初形成的cccDNA在肝细胞中是稳定的,而不需要在体外感染系统中持续补充,并且不需要从头含DNA的核衣壳的贡献。因此,衣壳再导入的短期治疗靶向可能是消除慢性感染肝细胞中cccDNA的低效策略。
■乙型肝炎病毒可以在患者的一生中维持在肝脏中,导致肝损伤和癌症。我们已经确切地阐明了它是如何在被感染的细胞中维持自身的。这意味着我们对如何靶向病毒和治疗慢性感染有了更好的想法。
UNASSIGNED: Chronic HBV infection cannot be cured by current therapeutics owing to their limited ability to reduce covalently closed circular (ccc)DNA levels in the livers of infected individuals. Therefore, greater understanding of the molecular determinants of cccDNA formation and persistence is required. One key issue is the extent to which de novo nucleocapsid-mediated replenishment (reimport) contributes to cccDNA levels in an infected hepatocyte.
UNASSIGNED: We engineered an infectious HBV mutant with a genome encoding a stop codon at position T67 in the HBV core open reading frame (ΔHBc HBV). Importantly, ΔHBc HBV virions cannot initiate nucleocapsid synthesis upon infection. Long-term in vitro HBV infection markers were followed for up for 9 weeks in HepG2-NTCP cells (A3 clone) and HBV DNA was quantified using a newly-developed, highly-precise PCR assay (cccDNA inversion quantitative PCR).
UNASSIGNED: ΔHBc and wild-type (WT) HBV resulted in comparable expression of HBV surface antigen (HBsAg), which could be blocked using the entry inhibitor Myrcludex B, confirming bona fide infection via the receptor sodium taurocholate cotransporting polypeptide (NTCP). In primary human hepatocytes, Huh7-NTCP, HepG2-NTCP, and HepaRG-NTCP cells, comparable copy numbers of cccDNA were formed. cccDNA levels, transcription of viral RNA, and HBsAg secretion remained comparably stable in WT and ΔHBc HBV-infected cells for at least 9 weeks.
UNASSIGNED: Our results imply that de novo synthesised HBc plays a minor role in transcriptional regulation of cccDNA. Importantly, we show that initially-formed cccDNA is stable in hepatocytes without requiring continuous replenishment in in vitro infection systems and contribution from de novo DNA-containing nucleocapsids is not required. Thus, short-term therapeutic targeting of capsid-reimport is likely an inefficient strategy in eliminating cccDNA in chronically infected hepatocytes.
UNASSIGNED: The hepatitis B virus can maintain itself in the liver for a patient\'s lifetime, causing liver injury and cancer. We have clarified exactly how it maintains itself in an infected cell. This now means we have a better idea at how to target the virus and cure a chronic infection.